The goal of this project is to understand how DNA repair mechanisms contribute to genome stability within the heterochromatic regions of the genome. Replication, repair and recombination of repeated DNAs can result in chromosome rearrangements and other types of genome instability, which are associated with cancer progression and other human diseases. Using the Drosophila model system, we have shown that 1) chromatin proteins required for heterochromatin establishment and maintenance are required to preserve the stability of repeated sequences, 2) heterochromatin undergoes a dramatic expansion immediately after irradiation, 3) repair of damage in heterochromatin requires the homologous recombination (HR) pathway, and 4) early steps in HR repair occur within the heterochromatin domain, but late events only occur after foci associated with repeated sequences translocate outside the heterochromatin domain. Our working hypothesis is that the potentially damaging consequences of HR repair of repeated sequences are avoided by the unusual spatial and temporal dynamics of heterochromatin repair, which are mediated by heterochromatin components. We propose to use Drosophila animals and tissue culture cells to identify the molecules and mechanisms that regulate the response to DNA damage in heterochromatic, repeated sequences. Specifically, we will use a combination of live and fixed cell imaging, mutant analysis, protein biochemistry, and genomic approaches to determine how heterochromatin expansion, repair foci dynamics, and homologous recombination are regulated at repeated DNAs, and how these events contribute to genome stability. The results of these studies will greatly improve our understanding of how the heterochromatin environment ensures the stability of repeated sequences, which has important applications to the diagnosis and treatment of human diseases. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Human cancers contain massive numbers of chromosome rearrangements and other types of genome instability. Repeated sequences compose a large percentage of the fly and human genomes, and pose significant problems to the maintenance of genome stability. The results of this project will provide key information about how chromatin regulates the stability of repeated sequences, which will be important to the development of cancer diagnostic and treatment tools.